2

G3, 2021, 11(1), jkaa009

DOI: 10.1093/g3journal/jkaa009
Advance Access Publication Date: 27 November 2020
Investigation

Fast and inexpensive whole-genome sequencing library

preparation from intact yeast cells
Sibylle C. Vonesch ,1,* Shengdi Li ,1 Chelsea Szu Tu,1,† Bianca P. Hennig,1,‡ Nikolay Dobrev,2and Lars M. Steinmetz1,3,4,*
1
Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg 69117, Germany
2
European Molecular Biology Laboratory (EMBL), Protein Expression and Purification Facility, Heidelberg 69117, Germany
3
Stanford Genome Technology Center, Stanford University School of Medicine, Palo Alto, CA 94304, USA
4
Department of Genetics, Stanford University School of Medicine, Palo Alto, CA 94304, USA
†
Present address: Centre for Genomic Regulation (CRG), Barcelona, 08003, Spain.
‡
Present address: Center for Digital Health, Berlin Institute of Health (BIH) and Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin,
Humboldt-Universität zu Berlin, Berlin, Germany, 10117, Berlin, Germany.

*Corresponding authors: Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, Heidelberg 69117, Germany. vonesch@embl.de
(S.C.V.); lars.steinmetz@embl.de (L.M.S.)

Abstract
Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now
possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing
libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase effi-
ciently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however,
the time and effort spent on genomic DNA isolation limit the practicability of sequencing large numbers of samples. Here, we describe a
highly scalable method for preparing high-quality whole-genome sequencing libraries directly from Saccharomyces cerevisiae cultures in
less than 3 h at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting lysed yeast sphero-
plasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do
not show any GC bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based
kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms
between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of
yeast strains in a single day at a low price. By adjusting individual steps of our workflow, we expect that our protocol can be adapted to
other organisms.

Keywords: yeast; WGS; Tn5; tagmentation

Introduction prevents its use in large-scale projects for most laboratories. We

Whole-genome sequencing is a powerful tool in genomics re-
search by providing an unbiased and comprehensive view of the
genetic alterations present in a cell. Genomic information is
instrumental for identifying mutations that underlie observed
phenotypes and for pinpointing any collateral damage that can
occur as a side product of mutagenesis. As sequencing costs
continue to drop, the cost and time for preparation of sequencing
libraries have become the major limiting factor for large-scale
genome sequencing experiments.
The most rapid and scalable library preparation methods use
a hyperactive variant of the Tn5 transposase that fragments
double-stranded DNA and ligates synthetic oligonucleotide
adapters required for Illumina sequencing in a 5-min reaction
(Adey et al. 2010) (Illumina). While the one-step tagmentation re-
action greatly simplifies library preparation workflows compared
to traditional, multistep methods, and scales to the parallel proc-
essing of hundreds of samples, the cost of commercial reagents
(Hennig et al. 2017) and others (Picelli et al. 2014) have previously
described a robust Tn5 transposase purification strategy and ac-
companying library preparation protocol that allows generating
sequencing libraries with comparable quality but at dramatically
reduced cost compared to commercial solutions. In addition to a
hyperactive Tn5 enzyme variant carrying the previously reported
missense mutations E54K (Zhou and Reznikoff 1997; Zhou et al.
1998) and L372P (Weinreich et al. 1994), which increase the DNA-
binding efficiency and reduce inhibitory effects on Tn5 activity,
respectively, we introduced a second Tn5 construct carrying an
additional amino acid substitution (R27S) in the DNA-binding do-
main (Hennig et al. 2017), which allows adjusting the fragment
size distribution based on enzyme concentration during tagmen-
tation.
With Tn5-based adapter insertion using homemade enzymes,
genomic DNA isolation becomes the major bottleneck limiting
the practicability of sequencing large numbers of genomes.

Received: September 07, 2020. Accepted: October 28, 2020

C The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America.
V
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
2 | G3, 2021, Vol. 11, No. 1
Analogous to colony PCR, where lysed bacterial cells or yeast
spheroplasts are added to a PCR reaction without prior genomic
DNA isolation, we hypothesized that yeast whole-genome
sequencing library preparation could be simplified by applying
tagmentation directly to cells. A similar strategy incorporating
heat-based lysis prior to tagmentation was successfully applied
for whole-genome (Adey et al. 2010) and plasmid (Hwang et al.
2019) sequencing of bacterial cells. In contrast to bacteria, the
yeast Saccharomyces cerevisiae has a rigid cell wall that prevents ef-
ficient heat-based lysis. To facilitate cellular lysis the lytic en-
zyme Zymolyase (Kitamura and Yamamoto 1972), which actively
degrades the cell walls of various yeasts, thereby exposing the
fragile spheroplast, is routinely used in genomic DNA extraction
(van Burik et al. 1998; Wilkening et al. 2013) and colony PCR
(Fuxman Bass et al. 2016) protocols. With a molecular weight of
138 kDa the active Tn5 dimer-adapter complex does not far ex-
ceed the size of average polymerase enzymes (90 kDa), such
that treatment with Zymolyase should provide sufficient access
to genomic DNA to Tn5. Furthermore, a similar method is used
for Tn5-based adapter insertion into accessible DNA in yeast
ATAC-seq (Schep et al. 2015) protocols.
Here, we report a simplified whole-genome sequencing library
preparation protocol. By skipping a conventional genomic DNA
isolation step our method enables preparing sequencing-ready li-
braries directly from overnight yeast cultures in less than 3 h at a
cost of 34 cents per sample, while scaling to the parallel process-
ing of hundreds of samples. In place of a lengthy genomic DNA
isolation step, we incubate saturated yeast cultures with
Zymolyase, followed by heat inactivation to inactivate
Zymolyase and lyse spheroplasts, and directly apply tagmenta-
tion to the resulting samples. A nucleosome release step prior to
enrichment PCR improves the evenness of genomic coverage.
Resulting libraries are comparable in quality to libraries proc-
essed from extracted genomic DNA with a commercially avail-
able Tn5-based kit (Nextera XT, Illumina). We demonstrate the
simplicity and unbiased nature of the method by investigating
CRISPR/Cas9 on- and off-target editing outcomes in a panel of
yeast strains, reliably detecting edited variants, and shared poly-
morphisms between strains. Direct preparation of sequencing li-
braries from yeast cultures without DNA isolation enables
massively scaled genome sequencing experiments that have so
far been hampered by the time and effort spent on genomic DNA
isolation. At 34 cents library preparation costs are approximately
5- to 10-fold lower compared to libraries prepared from gDNA
extracted using the most simple and scalable kit-based methods.
By adjusting steps like initial lysis to specific sample require-
ments, we anticipate that our protocol can be applied to other
microbial cells, mammalian cells, or tissues.
Materials and methods
Yeast strain and media
We used the well-characterized yeast strain YJM789 (Wei et al.
2007), a derivative of a yeast isolated from the lungs of an AIDS
patient with pneumonia, in all experiments. YJM789 contains ap-
proximately 60,000 single nucleotide polymorphisms (SNPs) with
respect to the S. cerevisiae reference genome. For all experiments,
we inoculated a YPAD culture directly from glycerol stock and
grew it to saturation overnight. For experiments with genomic
DNA, we extracted genomic DNA using Master Pure genomic
DNA extraction Kit (Epicentre) following the manufacturer’s
instructions. We confirmed quality by gel and quantified genomic
DNA yield using the Qubit high-sensitivity DNA assay.
Cell lysis and adjustment of cell numbers
For Zymolyase treatment, cells from overnight cultures were pel-
leted at 1000 g for 3 min and resuspended in 50 ml of 300 U/ml
Zymolyase 100-T (AMSBIO) solution, incubated at 37 C for 30 min
followed by 10 min at 95 to inactivate Zymolyase and lyse spher-
oplasts. About 1.25 ml of the solution was used for tagmentation.
Final desired cell number was adjusted prior to Zymolyase treat-
ment by measuring OD600 of a diluted overnight culture and cal-
culating sampling volume assuming 30 Mio cells per 1 ml OD 1
culture (based on value from https://bionumbers.hms.harvard.
edu/bionumber.aspx?&id¼100986&ver¼3). Sampling volume
was adjusted such that 1.25 ml of the 50 ml Zymolyase reaction
contained the desired number of cells.
Tn5 adapter complex assembly
Tn5 was expressed and purified as previously described (Hennig
et al. 2017). Tagmentation adapters were annealed as previously
described (Hennig et al. 2017) and thawed on ice. As Tn5 storage
buffer (20 mM Tris pH 7.4, 800 mM NaCl, 50% Glycerol) contains
relatively high amounts of salts we mixed 2 ml of Tn5 protein
(0.5 mg/ml) with 0.5 ml of each annealed adapter (70 mM stock)
and 8 ml of 20 mM Tris pH 7.5 for adapter loading. Providing
adapters in slight excess to Tn5 favors all Tn5 dimer molecules to
be occupied by two adapters, shifting the equilibrium to the fully
saturated Tn5-adapter complex. The mixture was incubated at
23 for 30–60 min at 300 rpm in a thermoshaker. For its use in tag-
mentation, we further diluted the complex using dilution buffer
(10 mM Tris pH 7.5, 150 mM NaCl) to the desired dilution factor.
Tagmentation with homemade Tn5
Unless indicated otherwise 1.25 ml of lysed cells were mixed with
1.25 ml 1:10 diluted, adapter-loaded Tn5R27S, E54K, L372P, and 2.5 ml
tagmentation buffer TB1 [10 mM MgCl2, 25% dimethylformamide
(DMF) (v/v), 10 mM Tris-HCl final concentrations, adjusted to pH
7.6 using acetic acid]. Tagmentation buffer without DMF was pre-
pared as a 2 solution and DMF was added fresh immediately be-
fore tagmentation for every experiment. Samples were incubated
on a preheated thermocycler block for exactly 3 min at 55 C after
which 1.25 ml 0.2% SDS was added immediately for neutralization,
and samples were incubated for 5 min at room temperature. For
library amplification, we added 1.25 ml each of indexed P5 and P7
primer (10 lM), 6.75 ml of 2 KAPA HiFi Ready mix (KAPA
Biosystems), and 0.75 ml of DMSO to 6.25 ml of tagmentation reac-
tion. Salt or Proteinase K (ProK) treated samples were purified us-
ing 1.8 volumes of AMPure XP (Beckman Coulter) beads prior to
PCR amplification. A gap-filling step followed by 12-cycle enrich-
ment PCR was performed as described previously (Hennig et al.
2017) and samples were purified using 1.8 volumes AMPure XP
beads and eluted in 10 ml nuclease-free water. We quantified li-
brary yield using the Qubit high-sensitivity DNA kit and evalu-
ated library quality on an Agilent Bioanalyzer (high-sensitivity
DNA assay).
Tagmentation with Nextera enzyme
Samples processed from extracted genomic DNA with Nextera
XT kit (Illumina) were prepared following kit instructions with
the following changes: We used 150 pg DNA as input and scaled
down reaction volumes such that we used 1=4 of indicated
reagents per reaction. For cell-based protocols with Nextera en-
zyme, tagmentation, inactivation, and library amplification were
performed following kit instructions and using commercial
reagents, but only using 1=4 of indicated reagents. Briefly, 1.25 ml

gDNA (100 pg/ml) or zymolyase-treated cells were mixed with 2.5
ml Tagment DNA Buffer (TD) and 1.25 ml Amplicon Tagment Mix
(ATM). Samples were incubated on a preheated thermocycler
block for exactly 3 min at 55 after which 1.25 ml Neutralize
Tagment Buffer (NT) was added and samples were incubated for
5 min at room temperature. Salt or ProK treated samples were
purified using 1.8 volumes of AMPure XP (Beckman Coulter)
beads prior to PCR amplification. For enrichment PCR, we added
3.75 ml Nextera PCR Master Mix (NPM) and 1.25 ml each of Index N
and Index S primers, and ran the NXT PCR program described in
the kit instructions. Amplified libraries were purified using 1.8
volumes AMPure XP beads and eluted in 10 ml nuclease-free wa-
ter. We quantified library yield using the Qubit high-sensitivity
DNA kit and evaluated library quality on an Agilent Bioanalyzer
(high-sensitivity DNA assay).
Nucleosome dissociation
We tested two nucleosome dissociation methods. For salt treat-
ment, 6.25 ml of tagmented library was incubated with 3.75 ml of
3 M NaOAc (final 1.125 M) at room temperature for 1 h. For ProK
treatment 6.25 ml of tagmented library was incubated with 1.75 ml
0.4 mg/ml ProK (diluted from 20 mg/ml stock) at 65 C for 30 min
after tagmentation, unless indicated otherwise. When we applied
nucleosome dissociation before tagmentation, we combined cells
for several samples using 10 ml of the spheroplast solution as in-
put and eluting in the same volume after bead purification.
Combined changes
One hundred microliters of a saturated overnight culture (no OD
measured) was pelleted and resuspended in 25 ml 300 U/ml
Zymolyase solution and incubated at 37 for 30 min, followed by
10 min at 95 . We used 1.25 ml of this solution as input for tag-
mentation, corresponding to approximately 1.2 Mio cells (assum-
ing 30 Mio cells in 1 ml OD 1 culture and saturation at OD 8).
Samples were processed with indicated dilutions of Tn5E54K, L372P
or Tn5R27S, E54K, L372P using indicated tagmentation buffers (TB1 or
TB2) and tagmentation temperatures (37 or 55 ). After tagmen-
tation with the corresponding enzyme and inactivation, samples
were incubated with ProK for 30 min at 50 followed by 15 min at
65 and stored at 20 before proceeding with magnetic bead
cleanup (1.8) and enrichment PCR. Final purification was done
with 0.8 AMPure XP beads. The final protocol is also described
in Supplementary File S1. All samples were pooled and paired-
end 150 bp reads were generated on an Illumina NextSeq plat-
form. TB1: 10 mM MgCl2, 25% DMF (v/v), 10 mM Tris-HCl final
concentrations, adjusted to pH 7.6 using acetic acid. TB2: 8 mM
MgCl2, 20% DMF (v/v), 16 mM Tris-HCl final concentrations, ad-
justed to pH 7.6 using acetic acid. Tagmentation buffer without
DMF was prepared as a 2 solution and DMF was added fresh im-
mediately before tagmentation.
Library preparation for on- and off-target editing
analysis
Sixteen randomly picked MAGESTIC (Roy et al. 2018)-edited yeast
strains were inoculated from glycerol stocks into 100 ml YPAD in a
96 well plate, and grown overnight at 800 rpm at 30 . Entire cul-
tures were pelleted at 1000 g for 3 min, pellets were resuspended
in 25 ml 300 U/ml Zymolyase solution and incubated at 37 for
30 min, followed by 10 min at 95 . 1.25 ml of the solution was
mixed with 2.5 ml TB2 and 1.25 ml of 1:10 diluted Tn5R27S, E54K,

L372P, and tagmentation was performed at 55 C for 3 min, fol-
lowed by inactivation with 1.25 ml 0.2% SDS for 5 min at room
temperature. For nucleosome dissociation, samples were
S. C. Vonesch et al. | 3
incubated with 1.75 ml 0.4 mg/ml ProK for 30 min at 50 followed
by 15 min at 65 and stored at 20 before proceeding with mag-
netic bead cleanup (1.8) and enrichment PCR. Final purification
was done with 0.8 AMPure XP beads. All samples were pooled
and paired-end 150 bp reads were generated on an Illumina
NextSeq platform.
Whole-genome sequence analysis
Each whole-genome sequencing dataset was down-sampled to
consistent numbers of reads for further comparisons, using bash
command “gunzip -c reads.fastq.gz j head -n N.” For comparisons
that involved both samples with 150 and 75 bp reads all reads
were trimmed to length 75 prior to downstream processing. 30
Transposon adapter sequences were trimmed off using cutadapt
(Martin 2011) and trimmed paired-end reads mapped to the
S288c yeast reference genome (sacCer3 R64.2.1) using bwa (Li and
Durbin 2009) (v0.7.17). The bam files containing all mapped reads
were sorted and PCR duplicates filtered using an embedded ver-
sion of picard toolkits in gatk v4.0 (McKenna et al. 2010). Base qual-
ity score recalibration (BQSR) was performed using “gatk
BaseRecalibrator” to detect and correct systematic errors in the
original base accuracy scores. Statistics for assessing mapping
qualities were generated using samtools (Li et al. 2009) (v1.9) with
its “flagstat” option. Insert size distribution was extracted using
the command “gatk CollectInsertSizeMetrics.” Variant calling
was performed using “gatk HaplotypeCaller” with the haploid op-
tion “-ploidy 1.” Low-quality variant calls were annotated and fil-
tered using “gatk VariantFiltration” with expression “QD < 2.0 | FS
> 60.0 | MQ < 40.0 | SOR > 3.0 | MQRankSum < 12.5|
ReadPosRankSum < 8.0”.
SNP-calling rate assessment
We used the set of variants called in a sample processed with the
commercial Nextera pipeline as gold standard (number of total
true SNPs), and determined true and false-positive SNP-calling
rates with each protocol variation. For any given sample X, the
true positive rate was defined as (number of correctly called
SNPs/number of total true SNPs), while the false-positive rate
was defined as (number of miscalled SNPs/number of total SNPs
in X).
Nucleosome occupancy and GC content bias
analysis
We used two datasets of yeast nucleosome mapping (Lee et al.
2007; Schep et al. 2015). For tiling-array data, nucleosome occu-
pancy was assessed by the log ratio of probe signals between
mono-nucleosomal DNA and total genomic DNA samples. For
ATAC-seq data, we calculated transposon insertion frequency
per base using pyatac (Schep et al. 2015) (integrated in nucleoatac).
We grouped base positions of the rDNA locus into four catego-
ries: 35S rRNA genes, external transcribed spacers, internal tran-
scribed spacers, and nontranscribed regions (NTS), according to
published gene annotations of the S288c reference genome (http://
sgd-archive.yeastgenome.org/sequence/S288C_reference/genome_
releases/S288C_reference_genome_R64-2-1_20150113.tgz).
GC content was calculated for the yeast autosomal genome
with a 500 bp sliding window stepped by 250 bp. We took the
mean value from overlapped windows to represent GC content
bias for single-base position. For correlation tests (coverage vs nu-
cleosome occupancy, coverage vs GC content), we randomly sub-
sampled 100,000 positions to reduce computational complexity.
4 | G3, 2021, Vol. 11, No. 1
Off-target variant analysis
For the analysis of 16 strains edited by MAGESTIC, read mapping
and variant calling were performed as described above, except:
(1) gvcf files were generated using “gatk HaplotypeCaller -ERC
GVCF” before genotyping in cohort mode; and (2) additional filters
were applied to exclude variants with low read depth (<0.1 
sample average base coverage) and variants with missing genotypes
in 2 samples. Mutant allele frequency (MAF) was calculated by
counting fraction of nonreference alleles after excluding missing
genotypes. For each private mutation (unique variants present in
single strain), we scanned for gRNA-like sequences in both strand
directions of a (60þX) bp (X equals variant length) window cen-
tered at variant position. We measured the Levenshtein edit dis-
tance between on-target sequence and its best match in the
(60þX) bp window.
Data of in vitro on- and off-target sequence pairs were down-
loaded from the CIRCLE-seq publication (Tsai et al. 2017) and edit
distance between each pair was calculated accordingly.
Data availability
The pETM11-Sumo3-Tn5 plasmids carrying either the Tn5E54K,
L372Por Tn5R27S, E54K, L372P construct are available upon request.
Supplementary File S1 contains detailed step-by-step instruc-
tions of the protocol using homemade Tn5-based and nucleo-
some dissociation by salt or ProK. Supplementary File S2 contains
Supplementary Figures S1–S8. Supplementary Table S1 contains
protocol conditions and associated insert sizes. Supplementary
Table S2 contains editing outcomes for edited strains.
Supplementary Table S3 contains genotype information for
edited strains. The sequencing data discussed in this manuscript
have been deposited on SRA and are accessible through the ac-
cession number SRP282188.
Supplementary material is available at figshare DOI: https://
doi.org/10.25387/g3.13187504.
Results
Initial comparison of libraries prepared from
genomic DNA vs intact cells
We hypothesized that tagmentation could be applied directly to
heat-treated yeast spheroplasts to simplify whole-genome se-
quencing library preparation by skipping genomic DNA isolation.
Figure 1 illustrates the extraction-free library preparation work-
flow using homemade Tn5R27S, E54K, L372P. Instead of extracting ge-
nomic DNA, cells from saturated overnight cultures are exposed
to Zymolyase treatment to digest the cell wall, incubated at 95
for inactivation of Zymolyase and lysis of spheroplasts, and the
Tn5 adapter complex is added directly to the sample (Figure 1).
As nucleosomes could provide a barrier for polymerase during li-
brary amplification, we compared two methods for nucleosome
dissociation: salt or ProK treatment. High salt concentrations pro-
mote dissociation by decreasing the attractive force between pos-
itively charged histone proteins and negatively charged DNA
(Stacks and Schumaker 1979; Yager et al. 1989) while ProK is a
broad-spectrum serine protease (Bajorath et al. 1988) used for
crosslink release and nucleosome digestion in MNase assays
(Yuan et al. 2005). We performed all experiments with the well-
characterized yeast strain YJM789 containing approximately
60,000 SNPs relative to the yeast reference genome (Wei et al.
2007). In an initial attempt, we used 240,000 cells corresponding
to approximately 3 ng genomic DNA as input for tagmentation.
For comparison, we performed extraction-free preparation with
the commercial Nextera enzyme, using buffers and reagents
from the kit (Nextera XT, Illumina). As a reference standard, we
prepared a library from 150 pg extracted YJM789 genomic DNA
using reagents from the kit (NXTMP). Paired-end 75-bp reads
were generated on an Illumina MiSeq platform and reads
mapped to the reference yeast genome (sacCer3 R64.2.1) using
bwa (Li and Durbin 2009).
A major concern when skipping genomic DNA isolation is
the reduced accessibility of genomic DNA to Tn5 and polymer-
ase, leading to a greater variation in coverage across the ge-
nome (coverage bias). To assess the impact on the distribution
of genomic coverage, we quantified the number of reads map-
ping to each position, as well as average genome coverage for
each sample. We used the ratio of per-base coverage to average
coverage to illustrate coverage bias—the closer this ratio is to 1,
the more evenly the base is covered relative to the rest of the
genome. As a secondary bias metric, we calculated the fraction
of the genome covered at least eightfold, reflecting a common
variant calling filter. To measure the impact of coverage varia-
tion on variant calling, we determined the fraction of true posi-
tive SNPs and the false-positive call rate with increasing
sequencing coverage using the extracted sample (NXTMP) as a
reference.
With the Nextera enzyme, libraries prepared from cells with-
out nucleosome dissociation (NA) were highly similar to librar-
ies prepared from extracted genomic DNA (Figure 2,
Supplementary Figure S1), and a ProK nucleosome dissociation
step after tagmentation did not provide an additional benefit.
Despite equimolar pooling, we obtained very low read numbers
for the sample with salt treatment and could not evaluate it
across the entire range, but at lower sequencing coverage salt
treatment seemed to negatively affect variant calling perfor-
mance (Figure 2B). With homemade Tn5R27S, E54K, L372P in con-
trast, both nucleosome dissociation methods improved
coverage (Figure 2, A and C, Supplementary Figure S1A) and
SNP-calling rate (Figure 2B, Supplementary Figure S1B) com-
pared to no treatment (NA) when applied after tagmentation.
Salt treatment resulted in libraries that were indistinguishable
in quality from the extracted sample. Insert size distribution
(median/mode) was similar for the extracted sample (NXTMP
115/70) and extraction-free samples with salt treatment, while
ProK treatment resulted in larger insert sizes for both Nextera
(150/96) and Tn5R27S, E54K, L372P (182/139) (Supplementary Figure
S1A, Table S1).
The native Tn5 transposase has an integration site prefer-
ence (Goryshin et al. 1998; Shevchenko 2002; Reznikoff 2008) and
studies have reported a mild GC bias in vitro (Green et al. 2012)
and in library preparation applications (Lan et al. 2015). To as-
sess sequence-dependent bias in coverage, we calculated nucle-
otide composition of the yeast reference genome in 500-bp
sliding windows. No correlation with GC content was apparent
for either the extracted sample or our extraction-free method
using the Nextera kit (Supplementary Figure S1C). The absence
of PCR-dependent GC bias, as reported previously (Adey et al.
2010), could be a result of using the GC tolerant KAPA HiFi poly-
merase (KAPA Biosystems). Salt-treated libraries prepared with
the extraction-free protocol and homemade Tn5R27S, E54K, L372P
showed a mild GC bias (Supplementary Figure S1C). Overall,
these initial experiments showed that high-quality whole-ge-
nome sequencing libraries can be generated without a genomic
DNA isolation step with both commercial and homemade trans-
posase enzymes.
 S. C. Vonesch et al. | 5

Cell wall digestion Tagmentation Nucleosome release

100 ul saturated yeast culture Tn5-adapter complex dimers

3 min 55°C

300U/ml Zymolyase Tn5 inactivation Proteinase K 1.125 M NaOAc

30 min 37°C 50°C 30 min OR 23°C 60 min

0.2% SDS 5 min RT
10 min 95°C 65°C 15 min

tagmented gDNA nucleosome-free, tagmented gDNA

lysed spheroplasts
Bead clean Amplification Bead clean
gap filling - 72°C 3 min

12 cycles
1.8X amplification 0.8X
AmPure XP AmPure XP

2X wash Elute 6.25 ul 2X wash Elute 10 ul

70% EtOH nuclease-free water P5-i5 i7-P7 70% EtOH nuclease-free water

amplified tagmented gDNA libraries

Qubit hsDNA, Bioanalyser

Figure 1 A simplified whole-genome sequencing library preparation workflow without genomic DNA isolation. Workflow of cell wall digestion and heat-
based lysis, gDNA tagmentation, nucleosome dissociation, and subsequent NGS library preparation for dual index (i5/i7) whole-genome sequencing is
depicted. The double-stranded part of the linker oligonucleotide is shown in gray with a yellow dot depicting the phosphorylated 30 end. The 50
overhangs serving as templates for the indexed P5 (dark blue) or P7 (orange) adapter primers are shown in blue and red, respectively.

Optimizing protocol parameters with homemade
Tn5
Tagmentation tends to generate libraries with shorter insert sizes
than methods using mechanical or enzymatic fragmentation
(Adey et al. 2010), which can be a limitation for applications re-
quiring longer inserts. Compared to Tn5-based standard library
preparation from genomic DNA, library preparation from cells
with ProK-mediated nucleosome dissociation consistently pro-
duced longer fragments, providing an opportunity to address this
need. To identify robust conditions for an efficient, extraction-
free workflow with our in-house transposase compatible with
longer insert sizes, we extensively varied different reaction steps.
To assess whether providing Tn5 with a nucleosome-free DNA
substrate would improve genome coverage, we applied ProK
treatment after Zymolyase incubation but prior to tagmentation.
Compared with treatment after tagmentation, we observed larger
variation in genome coverage and lower performance for other
quality metrics (Supplementary Figure S2) for both Nextera and
Tn5R27S, E54K, L372P. The overall worse performance could stem
from a higher loss of material during bead-based purification af-
ter ProK treatment, as intact genomic DNA might not bind and
elute as efficiently as smaller, tagmented genomic DNA, but we
did not investigate this further. With 65 at the higher end of the
permissible temperature range for ProK activity, we tested incu-
bation at 50 , and 50 followed by 15 min at 65 . Both variations
improved coverage bias (Supplementary Figure S3A) and to a
smaller degree variant calling (Supplementary Figure S3, B and C)
compared to incubation at 65 , but the 50 treatment by itself led
to reduced insert sizes (Supplementary Figure S3D, Table S1) and
reduced genomic coverage at lower sequencing depths
(Supplementary Figure S3E). A combined incubation at 50 fol-
lowed by 65 resulted in highest library quality while retaining
larger insert sizes.
For initial testing, we measured optical density and adjusted
cell number of individual cultures, which limits throughput for
parallel processing. To evaluate the robustness of the protocol to
variable input cell numbers, we processed samples starting from
240,000 to 5 million cells. We adjusted sampling volume from the
6 | G3, 2021, Vol. 11, No. 1

A Nextera Tn5R27S,E54K,L372P
1.5 1.5
Method
NA
Density

Density
1.0 1.0
ProK
Salt
0.5 0.5 NXTMP

C
0.0 0.0
−1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0
log(coverage / mean(coverage)) log(coverage / mean(coverage))

B Nextera Tn5R27S,E54K,L372P
100 100
%True SNPs called

%True SNPs called

80 80 Method
NA
60 60
ProK
Salt
40 40
NXTMP

20
0 10 20 30 40 50 0 10 20 30 40 50
X genome coverage X genome coverage

C
Nextera Tn5R27S,E54K,L372P
%genome of coverage >= 8

%genome of coverage >= 8

100 100

75 75 Method
NA
50 50
ProK

25 25 Salt
NXTMP
0 0
0 10 20 30 40 50 0 10 20 30 40 50
X genome coverage X genome coverage
Figure 2 Comparison of different nucleosome dissociation methods in extraction-free library preparation. Samples starting from 240,000 cells were
processed following the workflow depicted in Figure 1. After tagmentation nucleosomes were dissociated using salt (blue) or ProK (green). NA (salmon)
indicates the lack of a nucleosome dissociation step. NXTMP (black dashed line) is a library processed from 150 pg extracted genomic DNA with the
Nextera XT kit and serves as reference standard. (A) Coverage bias distribution (log scale), with bias calculated as coverage at a base divided by average
genome coverage. The salt condition is omitted for Nextera due to insufficient reads for this sample. (B) Fraction of YJM789 SNPs called as a function of
sequencing depth. The reference set of true positive SNPs (52,373 SNPs) is derived from variant calling on a sample prepared from extracted genomic
DNA with the Nextera XT kit (NXTMP). (C) Fraction of the genome covered at least 8 as a function of sequencing depth.

saturated culture such that 1.25 ml of the Zymolyase reaction
contained the desired number of cells, to prevent any changes in
the volumes of reactions. Libraries generated from 240,000,
500,000, and 1 million cells (corresponding to approximately 3, 6,
and 12 ng of input DNA) were highly similar in terms of coverage
bias (Figure 3, A and E), variant calling (Figure 3, C and D), and in-
sert size distributions (Figure 3D). The main effect of increasing
cell number was an increased library yield after enrichment PCR.
Increasing input cell number to 5 million cells (60-ng DNA), in
contrast, increased coverage bias and resulted in lower SNP-
calling rate, potentially due to an unfavorable ratio of Tn5 to
DNA molecules or insufficient lysis at higher cell densities. The
robustness of the protocol toward cell number variation facili-
tates parallel processing of many samples as it makes it unneces-
sary to measure optical density and adjust cell number of
individual samples.
Previous studies have reported that magnesium chloride and
DMF concentrations in the tagmentation buffer affect
 S. C. Vonesch et al. | 7

Tn5R27S,E54K,L372P Tn5R27S,E54K,L372P
A D
1.5 Cell number
0.0075 240K
500K

Density
Density

1.0
0.0050 1M
5M
0.5 0.0025 NXTMP

0.0 0.0000
−1.0 −0.5 0.0 0.5 1.0 0 200 400 600
log(coverage / mean(coverage)) Insert size (bp)

B E
Tn5R27S,E54K,L372P Tn5R27S,E54K,L372P

%genome of coverage >= 8

100 1.00
%True SNPs called

80 0.75

60 0.50

0.25
40

0.00
0 10 20 30 40 50 0 10 20 30 40 50
X genome coverage X genome coverage

C
15 Tn5R27S,E54K,L372P
%False postive calls

Cell number
10 240K
500K

5 1M
5M
NXTMP
0
0 10 20 30 40 50
X genome coverage
Figure 3 Extraction-free library preparation is robust to variation in input cell number. Samples starting from 240,000 (salmon), 500,000 (green), 1
million (turquoise), or 5 million (purple) cells were processed using homemade Tn5R27S, E54K, L372P and nucleosome dissociation by incubation with ProK
after tagmentation. Cell numbers between 240,000 and 1 million yield stable results in terms of library quality. (A) Coverage bias distribution (log scale),
with bias calculated as coverage at a base divided by average genome coverage. (B) Fraction of YJM789 SNPs called as a function of sequencing depth.
The reference set of true positive SNPs (52,373 SNPs) is derived from variant calling on a sample prepared from extracted genomic DNA with the
Nextera XT kit (NXTMP). (C) Fraction of false-positive SNP calls as a function of sequencing depth, with false-positive call rate ¼ number of miscalled
SNPs/total number of SNPs. (D) Distribution of fragment insert sizes. Final cleanup used 1.8 Ampure beads. (E) Fraction of the genome covered at least
8 as a function of sequencing depth. NXTMP (black dashed line) is a library processed from 150 pg extracted genomic DNA with the Nextera XT kit and
serves as reference.

performance (Picelli et al. 2014). As our tagmentation buffer (TB1:
10 mM MgCl2, 25% DMF and 10 mM Tris-HCl, pH 7.6) was opti-
mized for cDNA libraries, we varied buffer composition to im-
prove tagmentation of genomic DNA from lysed spheroplasts.
We used 500,000 cells in these tests and omitted the nucleosome
dissociation step to evaluate the effect of buffer changes only.
Lowering the DMF concentration to 20% led to a slight reduction
in coverage bias (Supplementary Figure S4, A and E) and im-
proved variant calling performance (Supplementary Figure S4, B
and C) to almost the same level as the extracted sample.
Lowering DMF further to 17.5%, 15%, or 10% (only 10% shown in
plot) had no clear benefit compared to 20% DMF. In addition to
DMF, we altered the concentration of both magnesium chloride
and Tris-HCl for an optimized tagmentation buffer TB2 contain-
ing 8-mM MgCl2, 20% DMF, and 16-mM Tris-HCl, pH 7.6 (final
concentrations).
As both altered ProK and tagmentation conditions led to small
but discernible improvements in library quality individually, we
8 | G3, 2021, Vol. 11, No. 1
next evaluated them in combination. Taking advantage of the ro-
bustness of the protocol to variations in input cell number, we
resuspended pellets of 100 ml saturated overnight cultures di-
rectly in 25 ml 300 U/ml Zymolyase solution, without measuring
the optical density of the culture, and used 1.25 ml of this solution,
corresponding to approximately 1.2 Mio cells, as input for tag-
mentation. After tagmentation and inactivation, we incubated
each sample with ProK for 30 min at 50 followed by 15 min at
65 , and stored samples at 20 before proceeding with magnetic
bead cleanup and enrichment PCR. As our two different home-
made Tn5 enzyme versions (Tn5E54K, L372P and Tn5R27S, E54K, L372P)
had previously shown different characteristics (Hennig et al.
2017), we compared their performance to address whether one
would be superior to the other. Compared with tagmentation in
buffer TB1, coverage and variant calling were clearly improved
with buffer TB2 for both transposase versions, resulting in librar-
ies indistinguishable in quality parameters from the sample pre-
pared from extracted genomic DNA (Figure 4, A–C,
Supplementary Figure S5B) while retaining longer insert sizes
(Supplementary Figure S5A, Table S1). No correlation with GC
content was apparent in extraction-free libraries prepared with
either Tn5R27S, E54K, L372P or Tn5E54K, L372P (Figure 4D). To assess
whether we could further modulate the insert size distribution
specifically toward longer inserts, we evaluated alternative tag-
mentation parameters. We had previously found that higher en-
zyme dilutions, which decrease the ratio of Tn5 to DNA
molecules, resulted in a progressive shift of the insert size distri-
bution toward larger inserts for libraries made from cDNA
(Hennig et al. 2017). We observed a similar effect for libraries pre-
pared with our extraction-free protocol. Using a fivefold higher
enzyme dilution, we could shift insert sizes from 158/112 (me-
dian/mode) to 211/186 (Supplementary Figure S6, Table S1). Tn5
transposase is typically used at 55 in library preparation applica-
tions. Lowering tagmentation temperature to 37 resulted in
larger insert sizes (192/149), indicating reduced activity at this
temperature, without affecting library quality (Supplementary
Figure S6). In summary, we identified optimized nucleosome dis-
sociation and tagmentation conditions, as well as strategies to
generate libraries with larger insert sizes than is common for
Tn5-based adapter insertion. We also demonstrated that our pro-
tocol is robust to variation in input cell numbers, which facili-
tates parallel processing of many samples.
Detailed characterization of libraries prepared
from genomic DNA vs intact cells
We extensively compared the genomic characteristics of libraries
generated with the extraction-free protocol, using Tn5R27S, E54K,
L372P processed libraries with TB2 as an example, with standard li-
brary preparation from extracted genomic DNA (NXTMP).
Coverage of individual positions across the genome was relatively
well correlated between standard and extraction-free methods.
Positions with high coverage in standard extraction-based library
preparation (NXTMP) tended to have high coverage in extraction-
free samples, with the notable exception of a population drop-
ping in coverage in the sample without genomic DNA extraction
(Supplementary Figure S7A). We observed the same trend for
extraction-free preparation using Nextera (with ProK treatment,
from Figure 2), indicating the coverage loss was specific to
extraction-free protocols and not related to the use of different
Tn5 enzymes. We mapped these positions to specific sites in the
yeast rDNA locus, which occurs in 100–200 tandem-arrayed
9.1 kb repeats on chromosome XII (Nomura et al. n.d.; Sandmeier
et al. 2002), making up almost 10% of the genome. The rDNA
locus exists in two distinct chromatin states (Merz et al. 2008), an
accessible and a highly condensed state, and only a small frac-
tion of the repeats is expressed in normal growth conditions,
which could explain the reduced coverage for these regions. This
is consistent with a higher variability in coverage across the 37S
rRNA gene of the locus specifically in extraction-free samples
(Supplementary Figure S7B). Across the whole genome, we ob-
served no correlation between coverage and nucleosome posi-
tions, as evaluated by ATAC-seq insertion frequency (Schep et al.
2015) (Supplementary Figure S7C) or nucleosome occupancy (Lee
et al. 2007) (Supplementary Figure S7D), and standard and
extraction-free samples again looked very similar. A few outlier
positions with distinctly higher coverage in the extraction-free
sample, specifically in regions with a nucleosome occupancy
score of zero (Supplementary Figure S7D), also mapped to the
rDNA locus, which shows a bias toward higher coverage of NTS
in extraction-free samples (Supplementary Figure S7B). Taken to-
gether, we conclude that our extraction-free protocol using
homemade Tn5 enzymes generates whole-genome sequencing li-
braries that are similar in quality to libraries prepared from
extracted genomic DNA using a commercial kit, at significantly
reduced cost and improved throughput and with the additional
benefit of generating longer inserts.
CRISPR-Cas9 on- and off-target activity profiling
in yeast
We applied our method to validate the presence of designed var-
iants and identify any unwanted off-target mutations in a set of
16 yeast strains edited with MAGESTIC, our pooled CRISPR guide-
donor method for massively parallel precision editing and geno-
mic barcoding (Roy et al. 2018). Unique DNA barcodes present at
the genomic barcode locus tag a designed variant in each strain
(Supplementary Table S2). We constructed sequencing libraries
of these 16 strains and performed 150PE sequencing, generating
an average of 20-fold coverage per genome. After read mapping,
variant calling, and variant filtering, we detected 131 SNPs and
145 small insertions or deletions across all samples (Figure 5A).
Of the 16 strains, 14 received the desired mutations while 2 car-
ried the wild-type allele at the targeted locus. Among the 276
called variants, 121 were background variants (MAF ¼ 1) repre-
senting the baseline genetic differences between the S288c-
derivative editing base strain and the S288c reference genome,
while 107 were private variants, uniquely present in single line-
ages (Supplementary Table S3).
Unintended mutations caused by Cas9 off-target activity
should be private mutations, given the uniqueness of each guide
RNA sequence and the dependence on sequence similarity to the
target (Doench et al. 2016). To assess the likelihood of each vari-
ant to be derived from an off-target cleavage event, we quantified
the sequence similarity of the window surrounding each private
mutation to the relevant target sequence (20 nt gRNA þ 3 nt
PAM). As a positive control set, we calculated the edit distances
between known on- and off-target sequence pairs captured by
CIRCLE-Seq (Tsai et al. 2017) and compared them to the spectrum
of edit distances in our dataset. There was a clear difference be-
tween the two datasets, with experimentally captured off-target
sites from CIRCLE-seq exhibiting edit distances of 0–6 to the tar-
get site, while all but one of our private variants showed edit dis-
tances of seven or higher (Figure 5B). Higher edit distances in the
CIRCLE-Seq data, corresponding to more mismatches in the off-
target relative to the on-target site, were associated with lower
cleavage activity (Supplementary Figure S8). This further sup-
ports that the observed private mutations were unlikely to be
 S. C. Vonesch et al. | 9

Tn5E54K,L372P Tn5R27S,E54K,L372P
A
1.5 1.5

Density
Density

1.0 1.0 tag buffer

TB1

0.5 0.5 TB2

NXTMP

0.0 0.0
−1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0
log(coverage / mean(coverage)) log(coverage / mean(coverage))

B Tn5E54K,L372P Tn5R27S,E54K,L372P
100 100
%True SNPs called

%True SNPs called

80 80
tag buffer
TB1
60 60
TB2

40 40 NXTMP

0 10 20 30 40 50 0 10 20 30 40 50
X genome coverage X genome coverage
C
Tn5E54K,L372P Tn5R27S,E54K,L372P
%genome of coverage >= 8
%genome of coverage >= 8

100
100

75 75
tag buffer
50 50 TB1
TB2
25 25
NXTMP

0 0
0 10 20 30 40 50 0 10 20 30 40 50
X genome coverage X genome coverage
D
Tn5E54K,L372P Tn5R27S,E54K,L372P

40 40
Coverage (fold)

Coverage (fold)

30 30
count count
250
20 200 20 200
150 150
100 100
50 50
10 10

0 0
0.2 0.3 0.4 0.5 0.6 0.7 0.2 0.3 0.4 0.5 0.6 0.7
GC content GC content
Figure 4 An extraction-free protocol using homemade enzymes produces libraries of the same quality as a commercial, extraction-based solution.
Samples were prepared from 100 ml saturated overnight culture with nucleosome dissociation by ProK treatment at 50 followed by 65 and
tagmentation buffer TB1 (salmon) or TB2 (turquoise), and with homemade Tn5E54K, L372P or Tn5R27S, E54K, L372P enzyme. (A) Coverage bias distribution
(log scale), with bias calculated as coverage at a base divided by average genome coverage. (B) Fraction of YJM789 SNPs called as a function of
sequencing depth. The reference set of true positive SNPs (52,373 SNPs) is derived from variant calling on a sample prepared from extracted genomic
DNA with the Nextera XT kit (NXTMP). (C) Fraction of the genome covered at least 8 as a function of sequencing depth. NXTMP (black dashed line) is a
library processed from 150 pg extracted genomic DNA with the Nextera XT kit and serves as reference standard. (D) GC content related coverage bias
extraction-free libraries. GC content of the S. cerevisiae reference genome was determined for 500-bp sliding windows with 250 bp overlap, and per-base
GC content was calculated as the average of overlapping windows.
10 | G3, 2021, Vol. 11, No. 1

A gRNA-targeted variant Genomic variant Additional variant alleles Missing values

16_AE2_I3
16_CB6_D11
16_DA5_B10
13_CG12_N23
16_CB12_D23
16_CB1_D1
16_CC6_F11
16_DC9_F18
16_DB2_D4
16_DF3_L6
16_DE7_J14
16_CC10_F19
16_CC5_F9
16_CA4_B7
16_CB10_D19
3_DF2_L4

0%
MAF

100%

0.6 CIRCLE-Seq captured

off-target sites
Density

0.4 private mutation sites

0.2

0.0
0 2 4 6 8 10 12
Edit distance
Figure 5 On- and off-target editing analysis in extraction-free libraries. (A) Overview of identified mutations in each edited strain (indicated on y-axis),
at a sequencing depth of 20. Designed target variants are shaded in blue. Additional genomic variants are indicated in red or orange, with orange sites
representing alternative, non-wild-type genotype calls at sites at which an alternative allele has already been detected, likely representing false-
positive variant calls. Sites at which no genotype information could be obtained (no coverage) are highlighted in gray. The x-axis depicts a histogram of
MAF of each variant. (B) Histogram of edit distances between sequence windows surrounding gRNA targeted variants and alternative variants,
reflecting sequence similarity between sites. Sequence similarity between experimentally captured CIRCLE-seq off-target variants (salmon) and their
respective gRNA targeted variant indicate expected edit distances if a variant is caused by Cas9-mediated off-target activity. Edit distances obtained for
private mutations in each strain in our dataset are depicted in turquoise.

caused by Cas9 off-target activity, but rather derived from spon-
taneous mutation events, accumulated in the cell divisions since
editing. In summary, we used our protocol to generate whole-
genome sequencing libraries directly from saturated cultures of
CRISPR-edited yeast strains, and detected designed on-target,
background, and spontaneous mutations in all strains. The fact
that the majority of the identified mutations were either com-
mon (MAF ¼ 1) or private, even at a moderate sequencing depth
of 20, indicates that our extraction-free method generated high-
quality, unbiased whole-genome sequencing libraries with even
coverage across the genome while substantially simplifying the
library construction process and reducing cost.
Discussion
We describe a simplified whole-genome sequencing library prep-
aration workflow to generate high-quality, sequencing-ready li-
braries directly from yeast cultures without genomic DNA
isolation. Enzymatic digestion of the yeast cell wall followed by
brief heat treatment of spheroplasts is sufficient for Tn5 to access
genomic DNA, and removal of residual nucleosomes prior to en-
richment PCR can improve the evenness of genomic coverage,
such that resulting libraries are indistinguishable in quality from
libraries prepared from extracted genomic DNA with a commer-
cial kit (Nextera XT). With our method, the time from saturated
yeast culture to library is reduced to less than 3 h, enabling mas-
sively scaled sequencing projects by eliminating the time- and
labor-intensive genomic DNA isolation step while preserving li-
brary quality. The robustness of the protocol toward input cell
number variation further facilitates parallel processing of many
samples as it makes it unnecessary to measure optical density
and adjust sampling volumes of individual cultures. In addition
to its simplicity and the benefits in throughput, the protocol is
highly affordable with reagent costs of 34 cents per sample when
using our homemade Tn5 enzymes (Hennig et al. 2017). This is an
approximately 5- to 10-fold reduction compared to samples pre-
pared from gDNA extracted using the most simple and scalable
kit-based methods. Combining our method with low-coverage

(3) sequencing allows genotyping of segregant panels or deter-
mining targeted genome mutagenesis outcomes for only $1 per
genome. We believe that these advances will enable massively
scaled genome sequencing experiments that have so far been
hampered by the time, effort, and cost spent on genomic DNA
isolation.
Depending on the individual needs and resources of a lab,
we present several options for extraction-free library prepa-
ration. If homemade Tn5 is not available, high-quality librar-
ies can be generated using the Nextera XT kit on zymolyase
and heat-treated cells, without a nucleosome dissociation
step. To save cost, it is sufficient to only use 1=4 of indicated
reagents. With either of our homemade Tn5 enzymes cost is
reduced to 34 cents per sample, and high-quality libraries
can be obtained by including a nucleosome dissociation step
by salt or ProK. Libraries generated by transposase-mediated
adapter insertion (tagmentation) tend to have shorter insert
size distributions compared with methods using mechanical
or enzymatic fragmentation. Our extraction-free method us-
ing ProK-mediated nucleosome dissociation generates longer
inserts than extraction-free preparation with no or salt-
mediated nucleosome dissociation and than the commercial
workflow on genomic DNA, without the need for size selec-
tion. We also identify additional tagmentation parameters
such as temperature and Tn5 to DNA ratio that allow flexible
modulation of insert size for custom applications. Insert size
distributions compatible with Illumina systems can be
obtained even with relatively high dilutions of the Tn5 en-
zyme, which further reduces cost of the assay.
With our method, we reliably detected designed on-target
and shared background mutations in a panel of edited yeast
strains. We could confidently call genotypes at all target sites
and detected the majority of mutations, representing differen-
ces due to shared genetic background, in all strains. This indi-
cates that our extraction-free method provides high-quality,
unbiased genomic information while substantially simplifying
the library construction process and reducing cost. While we
have only tested this workflow in yeast it should in principle be
transferable to other organisms by adjusting initial lysis condi-
tions to the cell type of choice. Adding a homogenization step
prior to lysis could further enable direct library preparation
from tissues.
Acknowledgments
We thank the EMBL Protein Expression and Purification facility
for production of the in-house Tn5 enzymes, and the EMBL
Genomics Core Facility for discussion and technical support. We
thank members of the Steinmetz group for discussions and sup-
port.
Funding
This work was supported by an Advanced Investigator grant from
the European Research Council (ERC) under the European
Union’s Horizon 2020 research and innovation programme (AdG-
742804—SystGeneEdit to L.M.S.). S.C.V. was supported by an
Advanced Postdoc Mobility Fellowship from the Swiss National
Science Foundation (grant number P300PA_177909).
Conflicts of interest: None declared.
S. C. Vonesch et al. | 11
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